Dengue, which is the most common arthropod-borne infection worldwide, affects at least 50 million people every year. (Teo et al., Transfusion Medicine 19:66-77 (2009)) The group of viruses responsible for causing Dengue fever and Dengue hemorrhagic fever is endemic in more than 100 countries, and consists of four antigenically related virus serotypes called DEN-1, DEN-2, DEN-3 and DEN-4 (i.e., Dengue-1, Dengue-2, Dengue-3 and Dengue-4, respectively). Despite extensive cross-reactivity among these viruses in serological tests, there is no cross-protective immunity in humans. Individuals living in an endemic area can have as many as four infections, one with each serotype, during their lifetimes. (Mackenzie et al., Nature Medicine Supplement 10:S98-S109 (2004)) Notably, nearly 2.5 billion people are at risk of infection with Dengue virus, and 500,000 hospitalizations are required each year as a result. (Teo et al., supra)
The Dengue viruses are the only known arboviruses that have fully adapted to humans. The principal mosquito vector, Ae. aegypti, is a highly domesticated insect that prefers feeding on humans, and laying eggs in artificial containers in and around houses. Ae. aegypti is an efficient epidemic vector of Dengue virus because it often feeds on, and thus transmits virus to, more than one individual in a single gonotrophic cycle. Secondary vectors of Dengue virus include Ae. albopictus and Ae. polynesiensis. Although the virus may be transmitted vertically from an infected female to her offspring, most mosquitoes become infected when they ingest blood from a person experiencing an accute infection. (Mackenzie et al., supra) The increased incidence of epidemic Dengue fever and Dengue hemorrhagic fever in the human population has been attributed to factors including: (1) increased population growth and urbanization, especially in tropical developing countries, (2) lack of effective mosquito control, including increased geographic distribution of Ae. aegypti, and (3) increased air travel which provides a means for transporting Dengue and other urban pathogens between population centers of the world. (Gubler, Clin. Microl. Rev. 11:480-496 (1998))
While other parts of the world may be more severely impacted, the emergence of Dengue-related disease as a major public health problem has been most dramatic in the American region. In 1970, only DEN-2 was present in the Americas, although DEN-3 may have had a focal distribution in Columbia and Puerto Rico. In 1977, DEN-1 was introduced and caused major epidemics throughout the region over a 16-year period. DEN-4 was introduced in 1981 and caused similar widespread epidemics. Also in 1981, a new strain of DEN-2 from Southeast Asia caused the first major DHF epidemic in the Americas (Cuba). DEN-3 virus recently reappeared in the Americas after an absence of 16 years. Indeed, there is a small, but significant, risk for Dengue outbreaks in the continental United States, which harbors two competent mosquito vectors (i.e., Ae. aegypti and Ae. albopictus) that are capable of transmitting Dengue viruses. (Gubler et al., Emerg Infect Dis 1:55-57 (1995)) Today all four serotypes are broadly distributed across virtually all regions of the world that harbor Dengue virus. (Mackenzie et al., supra)
Although the major route of transmission occurs through the Ae. aegypti mosquito vector, Dengue virus has also been transmitted through blood and organ transplantation. (Teo et al., supra) For example, transmission of Dengue infection has been reported from donor to recipient in one case of living donor renal transplant. Transmission during a bone marrow transplant was reported in one instance during a Dengue epidemic in Puerto Rico in 1994. One instance of transmission through blood transfusion involved a patient in Hong Kong who developed fever and other symptoms three days after a blood transfusion. The donor was asymptomatic at the time of donation but developed mild symptoms of Dengue fever one day after blood donation. An archived sample from the donation also tested positive for Dengue virus by RT-PCR. Another instance of transfusion-related illness involved the transmission of Dengue from an asymptomatic blood donor who developed an acute febrile illness the day after donating blood. Retrospective investigation confirmed Dengue infection in the recipients of the three blood products from his donation. Two recipients had Dengue fever with some evidence of capillary leakage, whereas the platelet recipient had asymptomatic seroconversion. All recovered without sequelae. A stored serum sample from the donation tested positive for DEN-2 by RT-PCR. (Teo et al., supra)
While there may be clear reason for wanting to detect all four Dengue serotypes, implementation of a single assay that is highly sensitive for all serotypes has been hampered by limited relatedness of the viral targets at the nucleic acid level. For example, Forattini in Dengue Bulletin 27:91-94 (2003), and Domingo et al., in Dengue Bulletin 28:87-95 (2004) have both presented phylogenetic trees showing that DEN-4 is highly diverged from the remaining three serotypes.
Previous attempts by others to create nucleic acid-based assays for detecting Dengue virus have met with some success. For example, Usawattanakul et al., in Dengue Bulletin 26:125-130 (2002), describe a transcription-based nucleic acid amplification assay able to detect all four Dengue serotypes in the 3′ region of the viral genome with a sensitivity equal to 1 PFU/ml (see Abstract). The authors present electrophoretic results indicating a graded decrease in the amount of amplification product synthesized at different input levels of the four Dengue serotypes. No amplification product was detected below 0.1 PFU/ml for any of the Dengue targets. Notably, the target-complementary sequences of the primers and probe employed by Usawattanakul et al., are substantially identical to oligonucleotide sequences disclosed in U.S. Pat. No. 6,333,6150.
Our own efforts to create a sensitive assay using the 3′ region of the viral genome as a target for amplification resulted in an assay having an approximately ten-fold improvement in sensitivity for all four serotypes. However, that assay was characterized by dramatically different sensitivities for the different serotypes at very low levels of input target. In our hands, 100% of DEN-2 was detected at a concentration of 0.001 PFU/ml, but DEN-1 was detected in only 20% of the cases. Accordingly, there remains a need for an amplified assay that is both highly sensitive, and similarly sensitive for all four Dengue virus serotypes.